As human immunodeficiency virus type-1 Protease (HIV-1-PR) is an essential enzyme in the viral life cycle, it is well known in the art that its inhibition can lead to a control of the acquired immune deficiency syndrome (AIDS).
The main properties inhibitory drugs must display are efficiency and specificity. Conventionally, this is achieved by either capping the active site of the enzyme (competitive inhibition) or through the binding to some other parts of the enzyme, hence provoking structural changes which make the enzyme unfit to bind the substrate (allosteric inhibition).
To the extent of our knowledge, all of the known HIV-1-PR inhibitors being available in the market such as, for instance, Indinavir or Sanquinavir, follow the former paradigm.
The great disadvantage in using such compounds in therapy is represented by the drug resistance that may be acquired and that could lead to escape mutants. As a consequence, the above drugs may loose their effectiveness due to the large production of virions in the cell, coupled with the error prone replication mechanism of retroviruses. Under the selective pressure exerted by known drugs, moreover, HIV-1-PR either mutates at the active site or at sites controlling its conformation, in such a way that the enzymatic activity is essentially retained, although the drug is not able to bind to its target anymore. The first signs of the failure of the drug therapy usually takes place 6-8 months after the starting of the treatment. Tomasselli A G and Heinrikson R L, Targetting the HIV-Protease in AIDS therapy: a current clinical perspective. Biochem. Biophys. Acta 2000; 1477: 189-214.
It thus remains of crucial importance to devise novel strategies to block the activity of HIV-1-PR.
Structural conformation, as an example, is a crucial feature in enzyme activities. In order to be active, in fact, the enzymes should stand and remain in, or anyway reach, their proper shape.
Therefore, feasible strategies in finding inhibitors may imply the inhibition of the folding process, known to be responsible of the protein shape, thus somehow preventing the protein to reach a final and active conformation.
A number of experimental and theoretical evidences suggests that globular, single-domain proteins avoid a time-consuming search in conformational space, folding through a hierarchical mechanism. To this extent, Ptitsyn and Rashin observed a hierarchical pathway in the folding of Mb [Ptitsyn O B and Rashin A A model of myoglobin self-organization. Biophys. Chem. 1975; 3: 1-20]. Lesk and Rose identified the units building the folding hierarchy of Mb and RNase on the basis of geometric arguments [Lesk A M and Rose G D. Folding units in globular proteins. Proc. Natl. Acad. Sci. USA 1981; 78: 4304-4308], deriving the complete tree of events which lead these proteins to the native state. Maity and coworkers found, through equilibrium and kinetic hydrogen exchange experiments, that the folding of cytochrome c, composed of five foldon units in the native conformation, proceeds by the stepwise assembly of the foldon units rather than through one amino acid at a time [Maity H, Maity M, Krishna M M G, Mayne L and Englander S W, The stepwise assembly of foldon units, Proc. Natl. Acad. Sci. USA 2005; 102: 4741-4746]. All of these studies describe a framework where small units composed of few consecutive amino acids build larger units which, in turn, build even larger ones, which eventually involve the whole protein [Baldwin R L and Rose G D, Is protein folding hierarchic? TIBS 1990; 24: 26-83]. The kinetic advantage of this mechanism is that, at each level of the hierarchy, only a limited search is needed for the smaller units to coalesce into the larger units belonging to the following level [Panchenko A R, Luthey-Schulten Z and Wolynes PG. Proc. Natl. Acad. Sci. USA 1995; 93: 2008-2013].
Lattice model calculations [Broglia R A and Tiana G., Hierarchy of Events in the folding of model proteins. J. Chem. Phys 2001; 114: 7267-7273; Tiana G and Broglia R A., Statistical Analysis of Native Contact Formation in the Folding of Designed Model Proteins. J. Chem. Phys. 2001; 114 2503-2507] have shown that the folding of a single domain monomeric protein follows, starting from an unfolded conformation, a hierarchical succession of events, namely: 1) formation of few (2-4) local elementary structures (LES, containing 20%-30% of the proteins amino acids) stabilized by few highly conserved, strongly-interacting, “hot” [G. Tiana, R. A. Broglia, H. E. Roman, E. Vigezzi and E. Shakhnovich, Folding and Misfolding of Designed Protein-like Folding and Misfolding of Designed Protein-like Chains with Mutations, J. Chem. Phys 1998, 108: 757] hydrophobic amino acids (≈8% of the proteins amino acids) lying close along the polypeptide chain; 2) docking of the LES into the postcritical folding nucleus [Abkevich V I, Gutin A M and Shakhnovich E I, Specific nucleus as the transition state for protein folding, Biochemistry 1994 33:10026-10031], that is the formation of the minimum set of native contacts which bring the system over the major free energy barrier of the whole folding process; 3) relaxation of the remaining amino acids on the native structure shortly after the formation of the folding nucleus.
The “hot” sites which stabilize the LES are found to be very sensitive to (non-conservative) point mutations. As most of the protein stabilization energy is concentrated in these sites, the mutation of one or two of them has a high probability of denaturing the native state. On the other hand, mutating any other site (“cold” sites, even those “cold” sites belonging to the LES) has in general little effect on the stability of the protein [G. Tiana, R. A. Broglia, H. E. Roman, E. Vigezzi and E. Shakhnovich, Folding and Misfolding of Designed Protein-like Folding and Misfolding of Designed Protein-like Chains with Mutations, J. Chem. Phys 1998, 108: 757; Broglia R A, Tiana G, Pasquali S, Roman H E, Vigezzi E, Folding and Aggregation of Designed Protein Chains, Proc. Natl. Acad. Sci. USA 1998, 95:12930].
Making use of the same model it has been shown that it is possible to destabilize the native conformation of a protein making use of peptides whose sequence is identical to that of the protein LES [Broglia R A, Tiana G and Berera R, Resistance proof, folding-inhibitor drugs, J. Chem. Phys. 2003 118:4754]. Such peptides interact with the protein, in particular with their complementary fragments in the folding nucleus, with the same energy and through the same highly conserved amino acids which stabilize the folding nucleus, thus competing with its formation.
Among the advantages of these folding inhibitors with respect to conventional ones, it is worth pointing out that it is unlikely to observe the protein developing resistance through mutations. In fact, the present inhibitor binds to a LES essentially through the “hot” and “warm” amino acids of these structures, and a protein cannot mutate a LES [G. Tiana, R. A. Broglia, H. E. Roman, E. Vigezzi and E. Shakhnovich, Folding and Misfolding of Designed Protein-like Folding and Misfolding of Designed Protein-like Chains with Mutations, J. Chem. Phys 1998, 108: 757] and, in any case, not those “hot” amino acids which are essential to stabilize it as well as to bind to the other LES so as to form the folding nucleus, under risk of denaturation.
In this respect it is worth noting that neutral mutations (e.g., hydrophobic-hydrophobic) of these hot amino acids are possible, as they do not change in any remarkable way the stability of the corresponding LES, nor the strength and specificity with which LES dock to form the folding nucleus.